Epstein-Barr virus (EBV) is a major human pathogen that has been causally implicated in a broad spectrum of disease (lymphoid and epithelial tumors, acute infectious mononucleosis, oral hairy leukoplakia and possibly certain autoimmune diseases). More than 90% of adults are infected worldwide. Prevention of virus transmission would have a major impact on international health; however, the complex life cycle and the presence of viral oncoproteins have greatly complicated efforts to develop an attenuated vaccine. The highly specific nature of the interaction between EBV and its cell surface attachment protein, CD21, suggests that a recombinant protein vaccine based on CD21 could prove very effective. However, although the EBV receptor (CR2/CD21) was identified almost 20 years ago, the structural basis for the interaction of CD21 with its EBV ligand gp350/220 remains unknown. The objective of the proposed research is to provide an understanding of the interaction between this human tumor virus and its cellular receptor, CD21, at atomic resolution. Such data will clearly be necessary for the design of optimal agents that can interrupt virus binding. Our analysis will reveal the epitopes most critical for EBV attachment and define structural changes that accompany binding. This information will provide a platform for rational design of blocking agents and of immunogens that stimulate a highly protective immune response to EBV. In preliminary work, we have determined a high-resolution crystal structure of the EBV-attachment region of CD21 and through functional experiments and molecular modeling we have identified key EBV-binding epitopes on CD21. However, no structural data at all is available for the major EBV glvcoprotein gp350/220, an unusual membrane antigen that lacks similarity to any previously described protein. We are well aware that without this information, this is a high-risk project. Therefore we seek exploratory funding (R21) to develop the requisite data and to proceed to crystallize the gp350/220:CD21 complex. In Specific Aim 1, we will pursue the crystallization and structure analysis of functional portions of the EBV attachment protein gp350. In Specific Aim 2, we will produce complexes with CD21 and EBV 350/220 for structural analysis (this may succeed in the absence of 1). In Specific Aim 3, (an aim independent of 1 and 2) we will determine whether cyclic peptides that mimic the CD21 epitopes implicated in gp350 interaction by our model can prevent virus attachment in vitro. If successful, future studies will test these ligands in vivo. Relevant receptor and ligand proteins will be expressed from Pichia (yeast) and mammalian cells. The techniques of molecular biology, protein expression and biochemistry, x-ray crystallography as well as mammalian cell culture and analysis (virus binding and infection assays) will be utilized. The research proposed here will define the structural requirements for productive cellular attachment of EBV and will evaluate strategies to intervene with this interaction, thereby providing a solid foundation for the long awaited design of immunogens and of drugs that can prevent EBV infection and spread.